Compensatory Role of Insulin in the Extinction but Not Reinstatement of Morphine-Induced Conditioned Place Preference in the Streptozotocin-Induced Diabetic Rats

Neurochemical Research - Insulin receptors are distributed in the whole brain, including different parts of the reward circuit that modulate dopamine as the primary neurotransmitter implicated in...     

The Polyploid State Plays a Tumor-Suppressive Role in the Liver

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The Methionine-aromatic Motif Plays a Unique Role in Stabilizing Protein Structure

Of the 20 amino acids, the precise function of methionine (Met) remains among the least well understood. To establish a determining characteristic of methionine that fundamentally differentiates it from purely hydrophobic residues, we have used in vitro cellular experiments, molecular simulations, quantum calculations, and a bioinformatics screen of the Protein Data Bank. We show that approximately one-third of all known protein structures contain an energetically stabilizing Met-aromatic motif and, remarkably, that greater than 10,000 structures contain this motif more than 10 times. Critically, we show that as compared with a purely hydrophobic interaction, the Met-aromatic motif yields an additional stabilization of 1–1.5 kcal/mol. To highlight its importance and to dissect the energetic underpinnings of this motif, we have studied two clinically relevant TNF ligand-receptor complexes, namely TRAIL-DR5 and LTα-TNFR1. In both cases, we show that the motif is necessary for high affinity ligand binding as well as function. Additionally, we highlight previously overlooked instances of the motif in several disease-related Met mutations. Our results strongly suggest that the Met-aromatic motif should be exploited in the rational design of therapeutics targeting a range of proteins.     

The C-Type Lectin Receptor Mincle Binds to Streptococcus pneumoniae but Plays a Limited Role in the Anti-Pneumococcal Innate Immune Response

The innate immune system employs C-type lectin receptors (CLRs) to recognize carbohydrate structures on pathogens and self-antigens. The Macrophage-inducible C-type lectin (Mincle) is a FcRγ-coupled CLR that was shown to bind to mycobacterial cord factor as well as certain fungal species. However, since CLR functions during bacterial infections have not yet been investigated thoroughly, we aimed to examine their function in Streptococcus pneumonia infection. Binding studies using a library of recombinantly expressed CLR-Fc fusion proteins indicated a specific, Ca²⁺-dependent, and serotype-specific binding of Mincle to S. pneumonia. Subsequent experiments with different Mincle-expressing cells as well as Mincle-deficient mice, however, revealed a limited role of this receptor in bacterial phagocytosis, neutrophil-mediated killing, cytokine production, and antibacterial immune response during pneumonia. Collectively, our results indicate that Mincle is able to recognize S. pneumonia but is not required for the anti-pneumococcal innate immune response.     

Interleukin-7, but Not Thymic Stromal Lymphopoietin,Plays a Key Role in the T Cell Response to Influenza A Virus

The immune response to viral infection is ideally rapid and specific, resulting in viral clearance and establishment of immune memory. Some viruses such as HIV can evade such responses leading to chronic infection, while others like Influenza A can elicit a severe inflammatory response with immune-related complications including death. Cytokines play a major role in shaping the appropriate outcomes to infection. While Interleukin-7 (IL-7) has a critical role in T and B cell development, treatment with IL-7 has recently been shown to aid the adaptive T cell response in clearance of chronic viral infection. In contrast, the IL-7-related cytokine thymic stromal lymphopoietin (TSLP) has a limited role in lymphocyte development but is important in the immune response to parasitic worms and allergens. The role for these cytokines in the immune response to an acute viral infection is unclear. IL-7 and TSLP share IL-7Rα as part of their heterodimeric receptors with the gamma common chain (γc) and TSLPR, respectively. We investigated the role of IL-7 and TSLP in the primary immune response to influenza A infection using hypomorphic IL-7Rα (IL-7Rα^449F) and TSLPR^−/− mice. We found that IL-7, but not TSLP, plays an important role in control of influenza A virus. We also showed that IL-7 signaling was necessary for the generation of a robust influenza A-specific CD4 and CD8 T cell response and that this requirement is intrinsic to CD8 T cells. These findings demonstrate a significant role for IL-7 during acute viral infection.     

Mycobacterium marinum MgtC Plays a Role in Phagocytosis but is Dispensable for Intracellular Multiplication

MgtC is a virulence factor involved in intramacrophage growth that has been reported in several intracellular pathogens, including Mycobacterium tuberculosis and Salmonella enterica serovar Typhimurium. MgtC participates also in adaptation to Mg²⁺ deprivation. Herein, we have constructed a mgtC mutant in Mycobacterium marinum to further investigate the role of MgtC in mycobacteria. We show that the M. marinum mgtC gene (Mma mgtC) is strongly induced upon Mg²⁺ deprivation and is required for optimal growth in Mg²⁺-deprived medium. The behaviour of the Mma mgtC mutant has been investigated in the Danio rerio infection model using a transgenic reporter zebrafish line that specifically labels neutrophils. Although the mgtC mutant is not attenuated in the zebrafish embryo model based on survival curves, our results indicate that phagocytosis by neutrophils is enhanced with the mgtC mutant compared to the wild-type strain following subcutaneous injection. Increased phagocytosis of the mutant strain is also observed ex vivo with the murine J774 macrophage cell line. On the other hand, no difference was found between the mgtC mutant and the wild-type strain in bacterial adhesion to macrophages and in the internalization into epithelial cells. Unlike the role reported for MgtC in other intracellular pathogens, Mma MgtC does not contribute significantly to intramacrophage replication. Taken together, these results indicate an unanticipated function of Mma MgtC at early step of infection within phagocytic cells. Hence, our results indicate that although the MgtC function is conserved among pathogens regarding adaptation to Mg²⁺ deprivation, its role towards phagocytic cells can differ, possibly in relation with the specific pathogen''s lifestyles.     

The Elg1 Clamp Loader Plays a Role in Sister Chromatid Cohesion

Mutations in the ELG1 gene of yeast lead to genomic instability, manifested in high levels of genetic recombination, chromosome loss, and gross chromosomal rearrangements. Elg1 shows similarity to the large subunit of the Replication Factor C clamp loader, and forms a RFC-like (RLC) complex in conjunction with the 4 small RFC subunits. Two additional RLCs exist in yeast: in one of them the large subunit is Ctf18, and in the other, Rad24. Ctf18 has been characterized as the RLC that functions in sister chromatid cohesion. Here we present evidence that the Elg1 RLC (but not Rad24) also plays an important role in this process. A genetic screen identified the cohesin subunit Mcd1/Scc1 and its loader Scc2 as suppressors of the synthetic lethality between elg1 and ctf4. We describe genetic interactions between ELG1 and genes encoding cohesin subunits and their accessory proteins. We also show that defects in Elg1 lead to higher precocious sister chromatid separation, and that Ctf18 and Elg1 affect cohesion via a joint pathway. Finally, we localize both Ctf18 and Elg1 to chromatin and show that Elg1 plays a role in the recruitment of Ctf18. Our results suggest that Elg1, Ctf4, and Ctf18 may coordinate the relative movement of the replication fork with respect to the cohesin ring.     

The C-Terminal Portion of an Archaeal Toxin,aRelE, Plays a Crucial Role in Protein Synthesis Inhibition

Mutations of amino acids in the C-terminal region of an archaeal toxin, aRelE, from Pyrococcus horikoshii were characterized with respect to protein synthesis inhibitory activity and 70S ribosome-binding activity. The results suggest that basic residues at the C-terminal region in aRelE play a crucial role both in 70S ribosome binding and in protein synthesis inhibition activities.     

The L Gene of J Paramyxovirus Plays a Critical Role in Viral Pathogenesis

J paramyxovirus (JPV) was first isolated from moribund mice with hemorrhagic lung lesions in Australia in the 1970s. Recent sequencing of JPV (JPV-LW) confirms that JPV is a paramyxovirus with several unique features. However, neither JPV-LW nor a recombinant JPV based on its sequence (rJPV-LW) caused obvious illness in mice. In this work, we analyzed a different JPV isolate (JPV-BH), which behaved differently from JPV-LW; JPV-BH grew more slowly in Vero cells and had less of a cytopathic effect on tissue culture cells but caused severe disease in mice. We have determined the whole genome sequence of JPV-BH. There were several nucleotide sequence differences between JPV-BH and JPV-LW, one in the leader sequence, one in the GX gene, and three in the L gene. The high sequence identity between JPV-BH and JPV-LW suggests that JPV-BH and JPV-LW are the same virus strain but were obtained at different passages from different laboratories. To understand the roles of these nucleotide sequence differences in pathogenicity in mice, we generated a recombinant JPV-BH strain (rJPV-BH) and hybrid rJPV-BH strains with sequences from the leader sequence (rJPV-BH-Le-LW), the GX gene (rJPV-BH-GX-LW), and the L gene (rJPV-BH-L-LW) of JPV-LW and compared their pathogenicities in mice. We have found that rJPV-BH-L-LW was attenuated in mice, indicating that nucleotide sequence differences in the L gene play a critical role in pathogenesis.     

Serum Thiols as a Biomarker of Disease Activity in Lupus Nephritis

Lupus Nephritis (LN) develops in more than half of the Systemic Lupus Erythematous (SLE) patients. However, lack of reliable, specific biomarkers for LN hampers clinical management of patients and impedes development of new therapeutics. The goal of this study was to investigate whether oxidative stress biomarkers in patients with SLE is predictive of renal pathology. Serum biochemical and oxidative stress markers were measured in patients with inactive lupus, active lupus with and without nephritis and compared to healthy control group. To assess the predictive performance of biomarkers, Receiver Operating Characteristic (ROC) curves were constructed and cut-offs were used to identify SLE patients with nephritis. We observed an increased oxidative stress response in all SLE patients compared to healthy controls. Among the several biomarkers tested, serum thiols had a significant inverse association with SLE Disease Activity Index (SLEDAI). Interestingly, thiols were able too aptly differentiate between SLE patients with and without renal pathology, and serum thiol levels were not affected by immunosuppressive drug therapy. The decreased thiols in SLE correlated significantly with serum creatinine and serum C3 levels. Further retrospective evaluation using serum creatinine or C3 levels in combination with thiol’s cutoff values from ROC analysis, we could positively predict chronicity of renal pathology in SLE patients. In summary, serum thiols emerge as an inexpensive and reliable indicator of LN, which may not only help in early identification of renal pathology but also aid in the therapeutic management of the disease, in developing countries with resource poor settings.     

GPR55, a G-Protein Coupled Receptor for Lysophosphatidylinositol,Plays a Role in Motor Coordination

The G-protein coupled receptor 55 (GPR55) is activated by lysophosphatidylinositols and some cannabinoids. Recent studies found prominent roles for GPR55 in neuropathic/inflammatory pain, cancer and bone physiology. However, little is known about the role of GPR55 in CNS development and function. To address this question, we performed a detailed characterization of GPR55 knockout mice using molecular, anatomical, electrophysiological, and behavioral assays. Quantitative PCR studies found that GPR55 mRNA was expressed (in order of decreasing abundance) in the striatum, hippocampus, forebrain, cortex, and cerebellum. GPR55 deficiency did not affect the concentrations of endocannabinoids and related lipids or mRNA levels for several components of the endocannabinoid system in the hippocampus. Normal synaptic transmission and short-term as well as long-term synaptic plasticity were found in GPR55 knockout CA1 pyramidal neurons. Deleting GPR55 function did not affect behavioral assays assessing muscle strength, gross motor skills, sensory-motor integration, motor learning, anxiety or depressive behaviors. In addition, GPR55 null mutant mice exhibited normal contextual and auditory-cue conditioned fear learning and memory in a Pavlovian conditioned fear test. In contrast, when presented with tasks requiring more challenging motor responses, GPR55 knockout mice showed impaired movement coordination. Taken together, these results suggest that GPR55 plays a role in motor coordination, but does not strongly regulate CNS development, gross motor movement or several types of learned behavior.     

师资队伍培养是国家精品课程建设的关键

精品课程是具有一流教师队伍、一流教学内容、一流教学方法、一流教材、一流教学管理等特点的示范课程。精品课程建设的关键是一流教师队伍的培养。何谓"一流教师"?一流的教师应具有宽宏的气度、高深的学术造诣、精湛的教学艺术、较高的人文素养和良好的心理素质。     

CaSRB9基因的克隆及其在酿酒酵母形态发生中的作用

白色念珠菌是一种重要的人体致病真菌 ,致病机制与其形态发生紧密相关。酿酒酵母Flo8因子在其形态发生中起重要作用 ,我们把白色念珠菌基因组DNA导入酿酒酵母flo8基因缺失株中 ,筛选能够互补 flo8侵入生长缺陷的基因 ,分离到了一个与酿酒酵母SRB9同源的新基因 ,命名为CaSRB9。该基因全长 4998bp ,编码一种16 6 5个氨基酸的蛋白质。在双倍体酿酒酵母中CaSRB9可以部分互补MAPK途径基因缺失株以及 flo8缺失株的菌丝生长缺陷 ;在单倍体酿酒酵母中表达能够互补 flo8缺失株的侵入生长缺陷 ,但在MAPK途径基因缺失株中不能形成侵入生长     

The Ubiquitin Proteasome System Plays a Role in Venezuelan Equine Encephalitis Virus Infection

Many viruses have been implicated in utilizing or modulating the Ubiquitin Proteasome System (UPS) to enhance viral multiplication and/or to sustain a persistent infection. The mosquito-borne Venezuelan equine encephalitis virus (VEEV) belongs to the Togaviridae family and is an important biodefense pathogen and select agent. There are currently no approved vaccines or therapies for VEEV infections; therefore, it is imperative to identify novel targets for therapeutic development. We hypothesized that a functional UPS is required for efficient VEEV multiplication. We have shown that at non-toxic concentrations Bortezomib, a FDA-approved inhibitor of the proteasome, proved to be a potent inhibitor of VEEV multiplication in the human astrocytoma cell line U87MG. Bortezomib inhibited the virulent Trinidad donkey (TrD) strain and the attenuated TC-83 strain of VEEV. Additional studies with virulent strains of Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV) demonstrated that Bortezomib is a broad spectrum inhibitor of the New World alphaviruses. Time-of-addition assays showed that Bortezomib was an effective inhibitor of viral multiplication even when the drug was introduced many hours post exposure to the virus. Mass spectrometry analyses indicated that the VEEV capsid protein is ubiquitinated in infected cells, which was validated by confocal microscopy and immunoprecipitation assays. Subsequent studies revealed that capsid is ubiquitinated on K48 during early stages of infection which was affected by Bortezomib treatment. This study will aid future investigations in identifying host proteins as potential broad spectrum therapeutic targets for treating alphavirus infections.     

The PICALM Protein Plays a Key Role in Iron Homeostasis and Cell Proliferation

The ubiquitously expressed phosphatidylinositol binding clathrin assembly (PICALM) protein associates with the plasma membrane, binds clathrin, and plays a role in clathrin-mediated endocytosis. Alterations of the human PICALM gene are present in aggressive hematopoietic malignancies, and genome-wide association studies have recently linked the PICALM locus to late-onset Alzheimer's disease. Inactivating and hypomorphic Picalm mutations in mice cause different degrees of severity of anemia, abnormal iron metabolism, growth retardation and shortened lifespan. To understand PICALM's function, we studied the consequences of PICALM overexpression and characterized PICALM-deficient cells derived from mutant fit1 mice. Our results identify a role for PICALM in transferrin receptor (TfR) internalization and demonstrate that the C-terminal PICALM residues are critical for its association with clathrin and for the inhibitory effect of PICALM overexpression on TfR internalization. Murine embryonic fibroblasts (MEFs) that are deficient in PICALM display several characteristics of iron deficiency (increased surface TfR expression, decreased intracellular iron levels, and reduced cellular proliferation), all of which are rescued by retroviral PICALM expression. The proliferation defect of cells that lack PICALM results, at least in part, from insufficient iron uptake, since it can be corrected by iron supplementation. Moreover, PICALM-deficient cells are particularly sensitive to iron chelation. Taken together, these data reveal that PICALM plays a critical role in iron homeostasis, and offer new perspectives into the pathogenesis of PICALM-associated diseases.     

Tail Autotomy Plays No Important Role in Influencing Locomotor Performance and Anti‐Predator Behavior in a Cursorial Gecko

We used the frog‐eyed sand gecko (Teratoscincus scincus) as a model system to evaluate the locomotor costs of tail loss, and to examine whether tailless geckos use alternative anti‐predator behavior to compensate for the costs of tail loss. Of the 16 field‐captured geckos, eight were used as experimental animals and the remaining ones as controls. Locomotor performance, activity level and anti‐predator behavior were measured for experimental geckos before and after the tail‐removing treatment. Control geckos never undergoing the tail‐removing manipulation were measured to serve as controls for the measurements taken at the same time for experimental geckos. Experimental geckos did not differ from controls in activity level before they underwent the tail‐removing manipulation, but became less active thereafter. The mean locomotor stamina of tailless geckos was reduced by about 30% of the mean value for tailed ones. However, as the maximum stamina predicted from the laboratory trials is seldom required in nature, we expect that the costs associated with the reduced locomotor stamina may be relatively minor in T. scincus. All other examined locomotor (overall speed, maximal speed and stride length) and behavioral (distance to refuge, approach distance and flight distance) traits were not affected by the tail‐removing manipulation. Overall, our results suggest that tail autotomy plays no important role in influencing locomotor performance and anti‐predator behavior in lizards where the tail has no direct role in locomotion but is used to direct predatory strikes away from the torso.